Somatic, germline and sex hierarchy regulated gene expression during Drosophila metamorphosis

Abstract

Background: Drosophila melanogaster undergoes a complete metamorphosis, during which time the larval male and female forms transition into sexually dimorphic, reproductive adult forms. To understand this complex morphogenetic process at a molecular-genetic level, whole genome microarray analyses were performed.

Results: The temporal gene expression patterns during metamorphosis were determined for all predicted genes, in both somatic and germline tissues of males and females separately. Temporal changes in transcript abundance for genes of known functions were found to correlate with known developmental processes that occur during metamorphosis. We find that large numbers of genes are sex-differentially expressed in both male and female germline tissues, and relatively few are sex-differentially expressed in somatic tissues. The majority of genes with somatic, sex-differential expression were found to be expressed in a stage-specific manner, suggesting that they mediate discrete developmental events. The Sex-lethal paralog, CG3056, displays somatic, male-biased expression at several time points in metamorphosis. Gene expression downstream of the somatic, sex determination genes transformer and doublesex (dsx) was examined in two-day old pupae, which allowed for the identification of genes regulated as a consequence of the sex determination hierarchy. These include the homeotic gene abdominal A, which is more highly expressed in females as compared to males, as a consequence of dsx. For most genes regulated downstream of dsx during pupal development, the mode of regulation is distinct from that observed for the well-studied direct targets of DSX, Yolk protein 1 and 2.

Conclusion: The data and analyses presented here provide a comprehensive assessment of gene expression during metamorphosis in each sex, in both somatic and germline tissues. Many of the genes that underlie critical developmental processes during metamorphosis, including sex-specific processes, have been identified. These results provide a framework for further functional studies on the regulation of sex-specific development.

Figure 1

The Drosophila melanogaster sex determination hierarchy. The Drosophila sex determination hierarchy consists of a cascade of sex-specific alternatively spliced pre-mRNAs culminating in the production of sex-specific transcription factors encoded by doublesex (dsx) and fruitless P1 (fru P1). The primary determinate of sex is the X chromosome to autosomal chromosome (A) ratio. In females (X:A = 1), Sex Lethal (SXL) is produced and regulates the splicing of the pre-mRNA of transformer (tra), resulting in the production of TRA. TRA acts in conjunction with constitutively produced TRA-2, and regulates the alternative splicing of the pre-mRNAs of dsx and fru P1, leading to the production of the female-specific protein DSX^F. In males (X:A = 0.5), SXL is not produced and dsx and fru P1 pre-mRNAs undergo default splicing, resulting in the production of the male sex-specific proteins DSX^M and FRU^M. In addition, in females SXL represses dosage compensation, the process by which transcription of genes on the single X chromosome in males is up-regulated to roughly equal that of the two X chromosomes in females.

Figure 2

The number of genes with somatic sex-differential transcript levels differs across metamorphosis. The abscissa indicates the five time points during metamorphosis examined (0, 24, 48, 72, and 96 hour APF). The ordinate indicates the number of somatic, sex-differentially expressed genes, as identified by F-tests, q < 0.15, for sex or sex-time interaction terms, and a t-test of the means (q < 0.15), on the gene expression data of male and female tud progeny. Genes with female- and male-biased expression are shown in grey and black, respectively.

Figure 3

Clusters of genes with similar expression profiles in somatic tissues during metamorphosis. Clusters were generated using gene expression data from male and female tud progeny at five time points during metamorphosis, indicated at top of clustergram. Expression profiles for each cluster were generated by averaging the gene expression data at each time point for every gene in the cluster in both sexes. Yellow and blue indicates high and low levels of expression compared to a common reference, respectively. To the right of the clustergram, black and grey indicates a cluster in which gene expression is at a peak (enriched) or at a trough (depleted), respectively, relative to the average expression value across all time points. Clusters are at a peak or trough of expression if average expression was 2/3 of a standard deviation above or below the mean expression value of the five time points, respectively. Functional annotation represents functional categories that were overrepresented among the genes in the cluster, as determined by the program DAVID (P < 0.05 [22]).

Figure 4

Expression profiles of genes expressed in the male and female germline during metamorphosis. Expression profiles were generated by averaging the gene expression data of all genes that have high expression in the male or female germline at each time point. The data for these genes for following genotypes were averaged separately: male tud progeny, female tud progeny, wild type (CS) males, and wild type (CS) females. Yellow and blue indicates high and low levels of expression compared to a common reference, respectively.

Figure 5

Cluster of global expression profiles for Drosophila transcripts across metamorphosis. Dendrogram shows the similarity across transcriptional profiles at five time points during metamorphosis (0, 24, 48, 72, and 96 hour APF) for (A) male tud progeny (blue) and female tud progeny (red) and (B) male tud progeny, female tud progeny, wild type (CS) males (green), and wild type (CS) females (violet). Hierarchical clustering and Pearson correlation distance measure was used to group experiments based on their global expression profile using all expression data for annotated genes from each array. Values on each node represent the confidence of the separation (approximately unbiased P value) derived using multiscale bootstrap resampling and the program Pvclust [63].